Reverse transcriptase



Reverse transcriptase (RT) or RNA-dependent DNA polymerase transcribes single-stranded RNA into double-stranded DNA. HIV-1 RT is from the human immunodeficiency virus and is a heterodimer of P66 and P51. The images at the left and at the right correspond to one representative RT structure, i.e. crystal structure of HIV-1 Reverse transcriptase (1hmv). P15 is its RNAse H domain. NNRTI are the non-nucleoside inhibitors of HIV-1 RT and NRTI are the nucleoide inhibitors. M-MLV is RT from the leukemia virus. Being the protein that gives their name to Retroviruses, Reverse Transcriptase is, in company of Protease and Integrase, the most important part of the protein system involved in the process of infection of viruses like HIV, MuLV and AMV, and has the unusual property of transcribing ssRNA into dsDNA going against the Central Dogma of Molecular Biology. Since its discovery in 1970, the study of its properties and mechanisms of action have been of high interest among the scientific community due to the unique properties that makes it an important medical target enzyme and important tool for genetic engineering applications like RT-PCR in the construction of cDNA libraries.

Reverse Transcriptase is one of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground. -- CBI Molecular Playground Model --

Structure
 This hand-like heterodimer protein that has an usual length of 1000 residues (560 in Chain A and 440 for B), the third of them involved in alpha helical and almost a quarter in beta sheets, showing &alpha;+&beta; secondary structure domains; Chain A has an usual weight of 66KDa whereas Chain B is around 51KDa, those monomers are derived from the same gene but p51 lacks the amino acids of one active site and has a different tertiary structure conformation compared with p66, for this reason is enzymatically inactive.

Function
As a RNA-dependent DNA Polymerase, is able to recognize the initial RNA, transcribe it to ssDNA, cleave the remaining RNA and then build up the dsDNA, to do this the protein has two active catalytic zones. Chain A has the Polymerase active site that consist of two finger-like domains, one of them recognizes the initial nucleic acid by h-bonds interactions with phosphate groups of the side chains, then both domains make a conformational change closing the recognition hole to allow the second domain with the support a Magnesium ion coordination system, begin the transcription process adding the specific DNA nucleotides; this change is allowed by a flexible zone between the two previous domains that is used as a common pharmaceutical target site in order to prevent this change and by this way inhibit the activity, but this one is the only zone of the Chain A that has non-conserved aminoacids giving the virus more drug resistance.  As the same rate that the polymerization process occurs, the other active site known as the Ribonuclease H domain, cleaves the RNA releasing the ssDNA that comes again trough the Polymerase active site to become dsDNA (all this with a second Magnesium coordinative system, that allows non-specific recognition, just with phosphates). Finally Chain B despite of the similar aminoacid sequence with Chain A has no enzyme activity, possibly its function is to stabilize and communicate both active sites, this varying the length between them in order to synchronize both functions. This seems to be the most general idea of the mechanism of action of Reverse Transcriptase, however the process remains unclear and new approaches are being reported. 

One of the principal issues about this protein comparative with the usual DNA polymerase (besides to the similarity with the Klenow fragment), is the lack of a correction mechanism (usually made by DNA PolIII in the DNA Polymerase) that increase the quota of errors, producing more mutations and by this way giving more facultative and resistance abilities to the virus.

3D Structures of Reverse transcriptase
Update June 2011

HIV-1 RT P66/P51
3dlk, 1hmv - P66/P51

1mu2, 1jle, 1hpz, 1hqu, 1qe1, 1dlo - P66/P51 (mutant)

1har - P66 N-terminal

3qip - P66/P51+NNRTI nevirapine + RNase H inhibitor

3lp0, 3lp1, 3lp2, 1vrt - P66/P51+NNRTI nevirapine

2hnd, 2hny, 1s1u, 1s1x, 1lw0, 1lwc, 1lwe, 1lwf, 1jlb, 1jlf, 1fkp - P66/P51 (mutant)+NNRTI nevirapine

3m8p, 3mec - P66/P51+NNRTI etravirine

3med - P66/P51 (mutant)+NNRTI etravirine

3mee, 2zd1- P66/P51+NNRTI rilpivirine

3meg, 2ze2, 3bgr - P66/P51 (mutant)+NNRTI rilpivirine

1ikw, 1fk9 - P66/P51 +NNRTI efavirenz

1jkh, 1ikv, 1fko - P66/P51 (mutant)+NNRTI efavirenz

3dya, 3e01, 3drp - P66/P51+NNRTI pyrazole

3drr, 3drs - P66/P51 (mutant)+NNRTI pyrazole

3irx, 3is9, 3lak, 3lal, 3lam, 3lan, 3ffi, 3di6, 3dle, 3dlg, 2rki, 3c6t, 3c6u, 2rf2, 2vg5, 2vg6, 2vg7, 2opp, 1jlq, 1ep4, 1c0t, 1c0u, 1dtt, 1dtq, 1bqm, 1rt1, 1rt2, 1rev, 1rtj, 1vru, 1hnv, 1hni - P66/P51+NNRTI

1s1t, 1s1v, 1s1w, 1s6p, 1s6q, 1s9e, 1s9g, 1suq, 1sv5, 3dm2, 3dmj, 3dok, 3dol, 2opq, 2opr, 2ops, 2ic3, 2hnz, 1jlg, 1bqn, 1uwb, 1tvr, 3qo9 - P66/P51 (mutant)+NNRTI

3kk1, 1c1b, 1rti - P66/P51+RTI

1jla - P66/P51 (mutant)+ NRTI

3ig1 - P66/P51+ inhibitor beta-thujaplicinol

3i0r, 3i0s, 3m8q, 3isn, 3ith, 1c1c, 3nbp - P66/P51+ pyrimidine derivative

1ikx - P66/P51 (mutant)+ pyrimidine derivative

1tv6 - P66/P51+ pyridine derivative

1jlc, 1iky, 1eet - P66/P51(mutant)+ pyridine derivative

2b5j, 2ban, 2be2, 1rth, 1hvt - P66/P51+ pyridone derivative

1lw2, 1rt3 - P66/P51 (mutant)+ NRTI

1tkt, 1tkx, 1tkz, 1tl1, 1tl3 - P66/P51+ quinoline derivative

2b6a - P66/P51+ THR-50

2i5j - P66/P51 (mutant)+hydrazone derivative

1rt4, 1rt5, 1rt6, 1rt7 - P66/P51+ carboxanilide derivative

1klm - P66/P51+piperazine derivative

2jle – P66+NNRTI

3kjv, 3kk3, 1r0a, 1n5y, 1n6q, 1hys - P66/P51+DNA

1j5o, 1rtd, 3klf - P66/P51 (mutant)+DNA

1hvu - P66/P51+RNA

1t03, 1t05 - P66/P51+DNA+Fab+NRTI

3jsm - P66/P51 (mutant)+DNA+NRTI

2hmi - P66/P51 (mutant)+DNA+Fab

2iaj - P66/P51 (mutant)+NRTI

3kk2 - P66/P51+DNA+NRTI

3jyt - P66/P51 (mutant)+DNA+NRTI

HIV-1 AZT-resistant RT P66/P51
3kli - P66/P51 (mutant)

3kle, 3klg, 3klh – P66/P51 (mutant) + DNA

HIV-1 RT P66
2jle – P66+NNRTI

1har - P66 N-terminal

HIV-1 RT P15
1rdh, 1hrh – P15

3lp3 – P15+inhibitor MK3

3k2p – P15+inhibitor beta-thujaplicinol

3hyf – P15+pyrimidine derivative

1o1w – P15+Mg - NMR

HIV-2 RT
1mu2 - RT (mutant)

Leukemia virus RT
2hb5, 1mml - catalytic fragment

2r2r, 2r2t, 2fjv, 2fjw, 2fjx, 1ztw, 2fvs, 2fvr, 2fvq, 2fvp, 1n4l – catalytic fragment+DNA

2r2s, 2r2u - catalytic fragment+DNA+Co Bleomycin

1ztt - catalytic fragment+DNA+netropsin

1rw3, 1nnd – fragment (mutant)

1i6j, 1d0e, 1d1u, 1qaj, 1qai – N-terminal+DNA

3fsi - catalytic fragment+DNA+trypanocidal

RT from telomerase
2b2a - RT telomerase catalytic subunit (mutant) - Tetrahymena thermophila

2r4g - RT telomerase RNA-binding domain

3du6, 3du5 - TcRT telomerase catalytic subunit - Tribolium castaneum

3kyl - TcRT telomerase catalytic subunit + DNA